Integrase is encoded by the Pol gene from the HIV provirus and can be expressed as an active recombinant protein. Our laboratory has pioneered the integrase inhibitors research field (PNAS 1993), discovered several families of lead inhibitors (Nature Rev Drug Discovery 2005;Current Topics in Medicinal Chemistry 2009;Viruses 2010) and patented some with the aim of therapeutic development. Our current studies are focused on the discovery of novel chemotype integrase inhibitors to overcome resistance to raltegravir and target novel site of integrase. In the past year we have reported novel chemotypes derived from Vpr peptides. These short peptides represent a novel kind of inhibitors. We have shown they could serve as therapeutics by the addition of a poly-arginine tail that confers cellular uptake and antiviral activity. They can also been used to build non-peptidic derivatives. We have also reported novel synthetic chemotypes affiliated with the diketo acid strand transfer inhibitors (phtalimide and quinolinonyl diketo acid derivatives) (collaboration with Dr. Terrence Burke, Laboratory of Medicinal Chemistry, CCR, NCI, and with Dr. Roberto DiSanto, University of Rome, Italy). We have developed a panel of recombinant integrase proteins bearing the mutations observed in patients that develop resistance to raltegravir and elvitegravir. We have compared for the first time raltegravir and elvitegravir and shown that both drugs are highly selective for the strand transfer reaction, while being more than 100-fold less potent against the 3-processing reaction, and almost inactive against the disintegration reaction mediated by integrase. The selective activity of raltegravir and elvitegravir against strand transfer (one of the 3 reactions mediated by integrase) demonstrates the very high specificity of the clinically developed strand transfer inhibitors. It is consistent with our pharmacological hypothesis (TIPS 2005) that the strand transfer inhibitors trap the integrase-viral DNA complex by chelating the divalent metals in the enzyme catalytic site following 3-processing of the viral DNA. We recently characterized in several publications the biochemical enzymatic activities and drug sensitivities of the integrase mutants that confer clinical drug resistance. Those same mutants are now available to test our novel inhibitors. We are expanding these studies to double-mutants in the integrase flexible loop that commonly arise in raltegravir-resistant patients. The working hypothesis is that the second mutation acts as gain of function to rescue the biochemical activity of integrase after it had become defective by the presence of the first mutation. One of aims is to understand the molecular mechanisms of such complementation and the structural connections between the flexible loop, the viral and host DNAs, and the inhibitors. Notably, we found that the flexible loop double-mutant 140S-148H is cross-resistant to both raltegravir and elvitegravir. On the other hand, the 143Y mutant is primarily resistant to raltegravir and minimally resistant to elvitegravir. These results provide a rationale for using elvitegravir in patients that develop resistance to raltegravir due to mutation 143Y (but not in the case of mutations 140S-148H). Having established the assays with a battery of recombinant mutant integrase, we are in a position to discover novel chemotypes to overcome raltegravir and elvitegravir resistance.